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| Status |
Public on Dec 24, 2023 |
| Title |
PA1_DRIP_RNaseH_minus_rep2 |
| Sample type |
SRA |
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| Source name |
PA1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: PA1
treatment: none
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| Treatment protocol |
At least 5×106 cultured cells were washed with DPBS, collected and resuspended into 1.6 mL 1×TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Then 83 μL 10% SDS was added to lyse cells at 37 ℃ for 1hr, and 20 μL 20 mg/mL proteinase K were added to digest proteins at 55 ℃ for at least 10hr.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Genomic DNAs (containing RNAs in the hybrid state) were extracted by phenol/chloroform extraction and ethanol precipitation gently. Then the extracted DNAs were fragmented by treatment with restriction enzymes cocktail (HindIII, EcoRI, BsrGI, XbaI, and SspI) overnight. Fragmented DNAs were recovered by phenol/chloroform extraction and ethanol precipitation gently, and dissolved into 200 μL water. Half of recovered DNAs were treated with RNase H enzyme (Thermo) to eliminate RNAs in the hybrids overnight at 37 ℃ as DRIP control sample, while the remaining half were treated with water as DRIP sample. Digested DNAs were extracted by phenol/chloroform extraction and ethanol precipitation gently, and dissolved into 100 μL water. 10 μg total DNAs in the DRIP sample or DRIP control sample was used for immunoprecipitation (IP) using 5μg S9.6 antibody (Karafast) overnight at 4 ℃, followed by incubation with 50 μL Dynabeads Protein G. The beads were then washed two times with binding buffer (100 mM NaPO4 pH 7.0, 1.4 M NaCl, 0.5% Triton X-100) and eluted by elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) with Proteinase K at 55 ℃ for 1hr. At last, DNAs in the DNA:RNA hybrids were extracted by phenol/chloroform extraction and ethanol precipitation for qPCR. For DRIP-seq, extracted DNAs were further digested with 0.1 mg/mL RNase A at 37 ℃ for 1 hr and recovered by phenol/chloroform extraction and ethanol precipitation.
Ribominus RNA libraries were prepared using Illumina TruSeq Stranded Total RNA LT Sample Prep Kit. All libraries were size-selected with AmpureXP beads (Agencourt) and quantified. using Agilent Bio analyzer 2100 and Qubit high-sensitivity dsDNA assay. Size-selected libraries were subjected to deep sequencing with Illumina NextSeq 500
1×100bp single-end Ribominus RNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
First filtered by using Trimmomatic v0.38; Next removed rRNA by bowtie v1.1.2;
Analyses by Bowtie2 v2.3.5; Duplicated reads were removed by Picard v2.22.1
Assembly: GRCh38/hg38 human reference genome with the GENCODE gene annotation (v28)
Supplementary files format and content: bigwig files include the mapping information
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| Submission date |
Dec 22, 2023 |
| Last update date |
Dec 24, 2023 |
| Contact name |
Li Yang |
| E-mail(s) |
liyang_fudan@fudan.edu.cn
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| Organization name |
Fudan University
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| Department |
Institutes of Biological Sciences
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| Street address |
131 Dong-An Road
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| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE183930 |
Linking circular intronic RNA degradation and function in transcription by RNase H1 |
| GSE251903 |
Detect R-loop signal by DRIP-seq in PA1 cell line |
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| Relations |
| BioSample |
SAMN39083260 |
| SRA |
SRX23007621 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7989698_PA1_DRIP_RNaseH_minus_rep2.picard.bw |
68.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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